Nucleic acid extraction apparatus and method of use thereof

ABSTRACT

The present invention discloses a nucleic acid extraction apparatus and a method using the apparatus for extracting nucleic acids and amplifying target nucleic acids. The nucleic acid extraction apparatus comprising: a nucleic acid extraction element, a waste storage chamber, and a reaction chamber, wherein the reaction chamber is selectively in communication with the nucleic acid extraction element and the waste storage chamber, and the apparatus realizes fluid exchange through the pressure between the waste storage chamber and the reaction chamber. In the present invention, by using an integrated control system, the conventional manual process of nucleic acid extraction and purification is integrated into a fully automatic closed process, making the operation more conveniently and quickly and improving the efficiency of experimental work.

CROSS REFERENCE TO RELATED APPLICATION

The present application claims the benefit of Chinese Patent ApplicationNo. 201811470025.2, filed on Nov. 26, 2018. The content of thisapplication including all tables, diagrams and claims is incorporatedhereby as reference in its entity.

FIELD OF THE INVENTION

The present invention relates to a field of biological detection, and inparticular to a nucleic acid extraction apparatus and a method using theapparatus for extracting nucleic acids and amplifying target nucleicacids.

BACKGROUND OF THE INVENTION

Nucleic acids are the basis for studies in molecular biology field.High-quality nucleic acids are essential for the studies of molecularmarkers, gene cloning and gene expressions, etc. As biological samples(such as blood, saliva, semen, or other secretions) are complex incomposition, generally it is required to carry out extraction,purification and amplification of target nucleic acids before subsequentstudies. At present, the existing nucleic acid extraction andamplification mainly have the following problems: first, due to largenumber of samples, complicated sample processing, nucleic acidextraction, purification and amplification steps, the manual operationsmay produce errors and make the operations to be more complicated, thusit is unable to perform efficient and rapid extraction and amplificationof target nucleic acids; second, most of molecular diagnostics need tobe carried out in the laboratory, and many grassroots units cannotestablish a standard molecular diagnostic laboratory, moreover,operators have different operating habits and proficiency, which maycause cross-contamination of samples during extraction and amplificationof nucleic acids; third, existing instruments and apparatus for nucleicacid extraction and PCR are often large in size and not suitable for useat sampling sites, which limits the applications of moleculardiagnostics to some extent. The automatic, closed and integratedoperation of nucleic acid extraction and amplification can shorten therelated process and reduce human influence and enhance the safety andeffectiveness of preparation of nucleic acid samples, and in addition,it can meet the requirements for small and portable apparatus for therapid detections in the grassroots units or on the sites.

U.S. Pat. No. 9,212,980 discloses an apparatus for nucleic acidextraction and amplification comprising a plurality of chambers, a fluiddisplacement region and a fluid processing region, wherein the fluidprocessing region is provided with a fluid processing material for cellcapture, cell lysis, binding analytes, etc., such as a filter. The fluiddisplacement region is used to temporarily store fluids and it is incommunication with the fluid processing region. During use, by adjustingthe position of a rotary valve, the fluid processing region isselectively in communication with the plurality of chambers, thus thefluid is driven to flow between the fluid processing region, the fluiddisplacement region and chambers by up and down movement of a piston. Inthis process, the fluid is controlled by a pair of ports of the valvebody. By rotating the valve body, the two ports selectively communicatewith the chambers in sequence to cause fluid displacement or movement,which may result in cross-contamination of samples in several channels,affecting the efficiency of DNA amplification. In addition, whenperforming DNA extraction, for example, biological cells are immobilizedin the fluid processing region by a fluid processing material such as afilter, and by pushing the piston down, the washing liquid and lysatesequentially flow through the fluid processing region to lyse thebiological cells and release the intracellular DNA. Due to the presenceof negative pressure in the fluid displacement region and the wastechamber and the clogging of the filter by the broken biological cellsand tissues, etc., the fluid is difficult to be discharged from thefluid displacement region when the piston and it is required to apply agreat push force, which is not easy to operate. Moreover, after thebiological cells are lysed, the intracellular DNA is mixed with the tinybiological tissues that cannot be adsorbed or filtered by the filter,and the lysate directly enters the reagent chamber to mix with theamplification reagents, resulting in insufficient purity of theextracted template DNA and adverse effect on PCR results.

SUMMARY OF THE INVENTION

In view of the shortcomings of the prior art, it is an object of thepresent invention to provide a nucleic acid extraction apparatus. Thepresent invention can realize convenient and miniaturized extraction ofDNA and achieve the purpose of functional integration, structuralminiaturization and automatic extraction of DNA through a microfluidicDNA extraction module.

In a first aspect, the present invention provides a nucleic acidextraction apparatus, comprising:

a nucleic acid extraction element, a waste storage chamber, and areaction chamber, wherein the reaction chamber is selectively incommunication with the nucleic acid extraction element and the wastestorage chamber, and the apparatus realizes fluid exchange through thepressure between the waste storage chamber and the reaction chamber.

As used herein, “fluid exchange” means that fluid can flow from oneplace to another. In the present invention, it means that fluid canenter a reaction chamber from a nucleic acid extraction element or awaste storage chamber, or can enter a nucleic acid extraction element ora waste storage chamber from a reaction chamber, to achieve repeatedlyswitch between these chambers.

As used herein, “selectively” means that the waste storage chamber isnot in fluidic communication with the reaction chamber when the reactionchamber is in fluidic communication with the nucleic acid extractionelement; and the reaction chamber is not in fluidic communication withthe nucleic acid extraction element when the waste storage chamber is influidic communication with the reaction chamber, that is, the reactionchamber is selectively in fluidic communication with the nucleic acidextraction element or in fluidic communication with the waste storagechamber when appropriate.

Preferably, for the apparatus, fluid enters the waste storage chamberfrom the reaction chamber by reducing the air pressure of the wastestorage chamber.

Preferably, the air pressure of the waste storage chamber for theapparatus is reduced by a venting element that is in communication withthe waste storage chamber.

Preferably, the venting element is a columnar suction column.

Preferably, the venting element is a cylindrical suction column.

Preferably, a gasket is disposed at the junction of the suction columnand the waste liquid storage chamber.

Preferably, a solid phase material for nucleic acid extraction isdisposed in the reaction chamber.

Preferably, the solid phase material for nucleic acid extraction is amagnetic bead.

When the solid phase material for nucleic acid extraction is a magneticbead, the reaction chamber has a permanent magnet, and the magnetic beadis fixed in the reaction chamber. The magnetic bead can specificallybind to free DNAs to form a magnetic bead-DNA complex; accordingly, DNAsare immobilized in the reaction chamber.

Preferably, the nucleic acid extraction element comprises:

a lysis chamber for adding and storing a mixture of sample and lysate;or,

a wash chamber for adding and storing washing liquid; or,

an eluent chamber for adding and storing eluent;

wherein, one or more of the lysis chamber, the wash chamber or theeluent chamber are in fluidic communication with the reaction chamber,respectively.

Preferably, the wash chamber comprises a primary wash chamber, asecondary wash chamber and a tertiary wash chamber, one or more of theprimary wash chamber, the secondary wash chamber or the tertiary washchamber being in fluidic communication with the reaction chamber,respectively.

It is to be understood that the apparatus described herein may comprisea plurality of wash chambers, for example, two, three, four, five, etc.,to satisfy nucleic acid purification using a plurality of differentwashing liquids, or to use a larger amount of the same washing liquid toperform washing, to improve the purity of nucleic acids.

Preferably, the apparatus may further comprise a nucleic acidamplification element, and the nucleic acid amplification element is aPCR reaction solution chamber that is in fluidic communication with thereaction chamber, inside of which is provided with reagents for a PCRreaction.

Preferably, the nucleic acid amplification element further comprises aPCR reaction tube that is in fluidic communication with the PCR reactionsolution chamber.

Preferably, the sidewall of the PCR reaction solution chamber isprovided with an input port and a discharge port that are in fluidiccommunication with the PCR reaction tube.

Preferably, the PCR reaction solution chamber comprises a primary PCRreaction solution chamber and a secondary PCR reaction solution chamber,wherein the primary PCR reaction solution chamber is in fluidiccommunication with the reaction chamber, wherein an input port isdisposed at the bottom of the sidewall of the primary PCR reactionsolution chamber, and a discharge port disposed at the top of thesidewall of the secondary PCR reaction solution chamber. The mixture ofthe PCR reaction solution and the nucleic acids enters the PCR reactiontube from the input port at the bottom of the sidewall of the primaryPCR reaction solution chamber. As the liquid enters, the air in the PCRreaction tube is discharged from the discharge port at the top of thesidewall of the secondary PCR reaction solution chamber, so as to allowthe mixture to enter smoothly.

Preferably, the apparatus comprises a microfluidic channel for fluidiccommunication with a reaction chamber, a waste storage chamber, anucleic acid extraction element or a nucleic acid amplification element.

Preferably, the microfluidic channel is radially extended on a rotarydisk, and the reaction chamber, the waste storage chamber, the nucleicacid extraction element or the nucleic acid amplification element are incommunication with or not in communication with each other by therotation of the rotary disk.

Preferably, the fluid exchange is achieved by a microfluidic channel.

Preferably, wherein the waste storage chamber is not in fluidiccommunication with the reaction chamber when the reaction chamber is influidic communication with the nucleic acid extraction element; and thereaction chamber is not in fluidic communication with the nucleic acidextraction element when the waste storage chamber is in fluidiccommunication with the reaction chamber.

Preferably, wherein the apparatus comprises a piston, the fluid in thenucleic acid extraction element enters the reaction chamber by themovement of the piston.

Preferably, wherein the movement of the piston is an upward movementalong the reaction chamber.

Preferably, the apparatus further comprises a venting element thatcauses the waste storage chamber to generate a negative pressure,thereby allowing the fluid in the reaction chamber to enter the wastestorage chamber.

Preferably, wherein the negative pressure drives the piston to movedownwardly along the reaction chamber.

Preferably, wherein the venting element that causes the waste storagechamber to generate a negative pressure is a suction column connected tothe waste storage chamber.

In a second aspect, the present invention provides a method forextracting a nucleic acid and amplifying a specific target nucleic acidusing a portable apparatus.

Preferably, the method comprises the following steps:

(1) providing a nucleic acid extraction apparatus, wherein the apparatuscomprising:

a nucleic acid extraction element for extraction of nucleic acids;

a waste storage chamber for storing waste liquid during the reaction;

a reaction chamber, being selectivity in communication with the nucleicacid extraction element and the waste storage chamber;

(2) allowing the reaction chamber to be in fluidic exchange with thewaste storage chamber or the nucleic acid extraction element to achieveextraction of nucleic acids.

Preferably, the fluid is exchanged between the reaction chamber, thewaste storage chamber, or the nucleic acid extraction element by apressure change between the waste storage chamber and the reactionchamber.

Preferably, the fluid is allowed to enter the waste storage chamber fromthe reaction chamber by reducing the air pressure in the waste storagechamber.

Preferably, the air pressure of the waste storage chamber is reduced bya venting element that is in communication with the waste storagechamber.

Preferably, the venting element is a cylindrical suction column.

Preferably, a solid phase material for nucleic acid extraction isprovided in the reaction chamber to bind the nucleic acid in the sampleto the solid phase material for nucleic acid extraction.

Preferably, the solid phase material for nucleic acid extraction is amagnetic bead.

Preferably, the nucleic acid extraction element comprises:

a lysis chamber for adding and storing a mixture of sample and lysate;or,

a wash chamber for adding and storing washing liquid; or,

an eluent chamber for adding and storing eluent;

wherein, one or more of the lysis chamber, the wash chamber or theeluent chamber are in fluidic communication with the reaction chamber,respectively.

Preferably, the wash chamber comprises a primary wash chamber, asecondary wash chamber and a tertiary wash chamber, one or more of theprimary wash chamber, the secondary wash chamber or the tertiary washchamber being in fluidic communication with the reaction chamber,respectively.

Preferably, the method further comprises a step of nucleic acidamplification, and the reaction chamber is in fluidic communication withthe nucleic acid amplification element to achieve nucleic acidamplification, wherein, the nucleic acid amplification element is a PCRreaction solution chamber that is in fluidic communication with thereaction chamber, inside of which is provided with reagents for a PCRreaction.

Preferably, the nucleic acid amplification element further comprises aPCR reaction tube that is in fluidic communication with the PCR reactionsolution chamber, to allow fluid to enter the PCR reaction tube foramplification reaction.

Preferably, the PCR reaction tube achieves fluidic communication throughan input port and a discharge port disposed on sidewall of the PCRreaction solution chamber.

Preferably, the PCR reaction solution chamber comprises a primary PCRreaction solution chamber and a secondary PCR reaction solution chamber,wherein the primary PCR reaction solution chamber is in fluidiccommunication with the reaction chamber, wherein an input port isdisposed at the bottom of the sidewall of the primary PCR reactionsolution chamber, and a discharge port disposed at the top of thesidewall of the secondary PCR reaction solution chamber.

Preferably, fluidic communication or exchange between the reactionchamber, the waste storage chamber, the nucleic acid extraction elementor the nucleic acid amplification element is achieved by a microfluidicchannel disposed on the nucleic acid extraction apparatus.

Preferably, the microfluidic channel is radially extended on a rotarydisk, and the reaction chamber, the waste storage chamber, the nucleicacid extraction element or the nucleic acid amplification element are incommunication with or not in communication with each other by therotation of the rotary disk.

Preferably, the fluid exchange is achieved by a microfluidic channel.

Preferably, wherein the waste storage chamber is not in fluidiccommunication with the reaction chamber when the reaction chamber is influidic communication with the nucleic acid extraction element; and thereaction chamber is not in fluidic communication with the nucleic acidextraction element when the waste storage chamber is in fluidiccommunication with the reaction chamber.

Preferably, wherein the fluid in the nucleic acid extraction elemententers the reaction chamber by the movement of a piston disposed on thereaction chamber.

Preferably, wherein the movement of the piston is an upward movementalong the reaction chamber.

Preferably, wherein the fluid in the reaction chamber enters the wastestorage chamber by a venting element that causes the waste storagechamber to generate a negative pressure.

Preferably, wherein the venting element that causes the waste storagechamber to generate a negative pressure is a suction column connected tothe waste storage chamber, and the negative pressure drives the pistonto move downwardly along the reaction chamber.

In a third aspect, the present invention provides a nucleic acidextraction apparatus. Preferably, the apparatus comprises a nucleic acidextraction element, a waste storage chamber, and a reaction chamber,wherein the reaction chamber is selectively in fluidic communicationwith the nucleic acid extraction element or the waste storage chamberthrough a microfluidic channel.

Preferably, the microfluidic channel comprises an inlet microfluidicchannel that allows the reaction chamber to be in fluidic communicationwith the nucleic acid extraction element, and a plurality of outletmicrofluidic channels that allow the reaction chamber to be in fluidiccommunication with the waste storage chamber.

Preferably, wherein the waste storage chamber is not in fluidiccommunication with the reaction chamber when the reaction chamber is influidic communication with the nucleic acid extraction element via theinlet microfluidic channel; and the reaction chamber is not in fluidiccommunication with the nucleic acid extraction element when the wastestorage chamber is in fluidic communication with the reaction chambervia the outlet microfluidic channel.

Preferably, the microfluidic channel is radially extended on a rotarydisk, and the reaction chamber, the waste storage chamber, the nucleicacid extraction element are in communication with or not incommunication with each other by the rotation of the rotary disk.

Preferably, the inlet microfluidic channel is different in length fromthe outlet microfluidic channel.

Preferably, the nucleic acid extraction element comprises: a lysischamber, a wash chamber or an eluent chamber, wherein one or more of thelysis chamber, the wash chamber or the eluent chamber being in fluidiccommunication with the reaction chamber via the inlet microfluidicchannel respectively.

Preferably, the wash chamber comprises a primary wash chamber, asecondary wash chamber and a tertiary wash chamber, wherein, one or moreof the primary wash chamber, the secondary wash chamber or the tertiarywash chamber being in fluidic communication with the reaction chambervia the inlet microfluidic channel respectively.

Preferably, the apparatus may further comprise a nucleic acidamplification element, and the nucleic acid amplification element is aPCR reaction solution chamber that is in communication with the reactionchamber, inside of which is provided with reagents for a PCR reaction.

Preferably, the PCR reaction solution chamber is in fluidiccommunication with the reaction chamber via an inlet microfluidicchannel.

In a fourth aspect, the present invention provides a nucleic acidextraction apparatus. Preferably, the apparatus comprises: a nucleicacid extraction element, a waste storage chamber, a nucleic acidamplification element, and a reaction chamber, wherein the nucleic acidamplification element comprises a PCR reaction solution chamber and aPCR reaction tube, and an input port and a discharge port are providedon the sidewall of the PCR reaction solution chamber for communicatingwith the PCR reaction tube.

Preferably, the PCR reaction solution chamber comprises a primary PCRreaction solution chamber and a secondary PCR reaction solution chamber,the input port is disposed at the bottom of the sidewall of the primaryPCR reaction solution chamber, and the discharge port is disposed at thetop of the sidewall of the secondary PCR reaction solution chamber.

Preferably, the input port is a through hole, which is in communicationwith a first port of a PCR reaction tube injection channel, forinjecting a fluid into a PCR reaction tube; the discharge port is athrough hole, which is in communication with a second port of a PCRreaction tube injection channel, for discharging the gas in the channel.

Preferably, the apertures of the input port and the discharge port aresmaller than the apertures of the first port and the second port.

Preferably, the apparatus is provided with a seal at the junction of thePCR reaction solution chamber and the PCR reaction tube.

Preferably, the seal has two through holes, wherein the size of thethrough hole connected between the seal and the PCR reaction solutionchamber corresponds to the size of the input port and the dischargeport, and the size of the through hole connected between the seal andthe PCR reaction tube corresponds to the size of the first port and thesecond port.

Preferably, the seal is made of an elastic material.

Preferably, the seal is made of silica gel.

Preferably, a non-absorbent material capable of slowly leaking gas isprovided at the top of the primary PCR reaction solution chamber.

Preferably, the non-absorbent material capable of slowly leaking gas ishigh-density hydrophobic cotton.

The present invention can achieve the following beneficial effects:

(1) The present invention adopts a reaction chamber having a hollowpiston and a hollow receiving chamber for extraction and purification ofnucleic acids in samples. The magnetic bead is fixed to a reactionchamber by a permanent magnet disposed in a hollow receiving chamber, sothat the nucleic acids released after lysis of samples are specificallyimmobilized in the reaction chamber, and with the up and down movementof the hollow piston, the integrated operation of nucleic acidextraction and purification can be realized. In the present invention, amagnetic bead is used as a solid phase material for nucleic acidextraction. When the permanent magnet is removed, the magnetic beadwhich binds the nucleic acid or the magnetic bead after the nucleic acidis eluted and dissociated will be scattered in the reaction chamber,which on one hand makes the extraction and purification process ofnucleic acid to be more convenient and controllable, and on the otherhand, the washing and replacement of solid phase material for nucleicacid extraction is more convenient.

(2) For the apparatus of the present invention, a venting element isprovided in the waste storage chamber for changing the air pressurebetween the waste storage chamber and the reaction chamber such that thewaste liquid in the reaction chamber is easily discharged; and byreducing air pressure by the venting element, the fluid in the reactionchamber is discharged into the waste storage chamber under the action ofnegative pressure, which needs not to pressurize the reaction chamber todischarge the liquid, facilitating the experimental operations.

(3) The apparatus and method of the present invention avoid the mixingof reagents caused by repeated microfluidic channels, improve the purityand concentration of the extracted DNA, thereby improving the efficiencyof the amplification reaction to achieve smooth and rapid reactions.

(4) The apparatus of the present invention achieves gas and liquidcommunication between the PCR reaction tube and the PCR reactionsolution chamber via the input port and the discharge port, effectivelyavoids the generation of bubbles during the liquid injection process,and moreover, through observation on the spill of liquid at thedischarge port, it can be judged whether the liquid injection iscompleted.

(5) In the present invention, by using an integrated control system, theconventional manual process of nucleic acid extraction and purificationis integrated into a fully automatic closed process, making theoperation more conveniently and quickly and improving the efficiency ofexperimental work.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are perspective cross-sectional views of a nucleic acidextraction apparatus of the present invention, wherein a partialenlarged view of FIG. 1B is a cross-sectional view of a piston.

FIG. 2 is a perspective view of a nucleic acid extraction apparatus ofthe present invention.

FIG. 3 is an exploded view of the apparatus of FIG. 2.

FIG. 4 is another perspective view of a nucleic acid extractionapparatus of the present invention.

FIGS. 5A and 5B are a perspective view of a suction column, wherein FIG.5A is a structural view of a suction column having a gasket, and FIG. 5Bis structural view of a suction column without a gasket.

FIG. 6 is another perspective view of a nucleic acid extractionapparatus of the present invention.

FIG. 7 is a top view of a nucleic acid extraction apparatus of thepresent invention.

FIG. 8 is another top view of a nucleic acid extraction apparatus of thepresent invention.

FIGS. 9A and 9B are a perspective view and a front view of a rotary diskof a nucleic acid extraction apparatus of the present invention.

FIG. 10 is a cross-sectional view of a rotary disk of a nucleic acidextraction apparatus of the present invention.

FIG. 11 is a cross-sectional view of a PCR reaction solution chamber ofa nucleic acid extraction apparatus of the present invention.

FIG. 12 is a structural diagram of a PCR reaction tube of a nucleic acidextraction apparatus of the present invention.

FIG. 13 is a perspective view of a base of a nucleic acid extractionapparatus of the present invention.

FIG. 14A is a front view and FIG. 14B is a rear view of a seal.

FIG. 15 is a structural diagram of a shell and a rotary disk of anucleic acid extraction apparatus of the present invention.

FIG. 16 is a perspective view of a nucleic acid extraction apparatusaccording to another embodiment of the present invention.

Notes: 1 top cover, 2 shell, 3 rotary disk, 4 base, 5 PCR reaction tube,6 suction column, 7 piston, 701 recess, 8 protrusion, 9 seal, 10magnetic bead, 11 permanent magnet, 201 reaction chamber, 2011 receivingchamber, 202 waste storage chamber, 203 lysis chamber, 204 wash chamber,2041 primary wash chamber, 2042 secondary wash chamber, 2043 tertiarywash chamber, 205 eluent chamber, 206 PCR reaction solution chamber,2061 Primary PCR reaction solution chamber, 2062 Secondary PCR reactionsolution chamber, 207 through hole, 208 input port, 209 discharge port,210 rib, 211 boss, 301 inlet microfluidic channel, 302 first outletmicrofluidic channel, 303 second outlet microfluidic channel, 304 thirdoutlet microfluidic channel, 305 fourth outlet microfluidic channel, 306opening, 307 boss, 501 first port, 502 second port, 601 gasket, 602suction column external end, 401 card slot, 402 card slot groove,901˜904 seal through hole, 905 groove.

DETAILED DESCRIPTION

The technical solutions in the embodiments of the present invention areexplicitly and completely described in the following with reference tothe accompanying drawings, which are only a part of rather than all ofthe embodiments of the present invention. All other technical solutionsobtained by those skilled in the art based on the embodiments of thepresent invention without creative work fall within the scope ofprotection of the present invention.

In the present invention, unless otherwise specified and defined,“connected”, “fixed, immobilized”, etc. should be understood broadly.For example, “fixed, immobilized” may be a fixed connection, or may be adetachable connection, or integrated; may be a direct connection, or anindirect connection through an intermediary, or may be an internalcommunication of two elements or an interaction relationship of twoelements, unless otherwise explicitly defined. For those skilled in theart, the specific meanings of these foregoing terms can be understood ona case-by-case basis in the present invention.

Example 1

FIGS. 1-4 show a particular embodiment of a nucleic acid extractionapparatus including a shell having a plurality of chambers. As seen inFIG. 2 and FIG. 3, the nucleic acid extraction apparatus has a top cover1 and a base 4, and the top cover 1 has openings for injecting samplesor reagents into the respective chambers.

FIG. 1 shows a nucleic acid extraction apparatus of the presentinvention, comprising a nucleic acid extraction element, a waste storagechamber 202 and a reaction chamber 201. The nucleic acid extractionelement is used to add or store reagents for nucleic acid extraction andpurification, the waste storage chamber 202 is used to store wasteliquid generated during the reaction process, and the reaction chamber201 is a place where nucleic acid is extracted. The nucleic acidextraction element, the waste storage chamber, and reaction chamber arelocated on the shell 2. In this embodiment, a reaction chamber isselectively in communication with the nucleic acid extraction elementand the waste storage chamber, and the apparatus achieves fluid exchangeby a pressure change between the waste storage chamber and the reactionchamber.

The term “fluid exchange” as used herein means that a fluid can flowfrom one place to another, and the flow process may be directed by somephysical structures. The term “directed by some physical structures”means that a fluid passively or actively flows to another place bypassing through the surface or interior space of these physicalstructures. The “passively” generally means that the flow is caused byan external force, for example, flow under a pressure. Specifically, inthe present invention, it means that a fluid can enter the reactionchamber from the nucleic acid extraction element or enter the nucleicacid extraction element or the waste storage chamber from the reactionchamber, to achieve repetitive switching between these chambers.

The term “selectively” as used herein means that, the waste storagechamber is not in fluidic communication with the reaction chamber whenthe reaction chamber is in fluidic communication with the nucleic acidextraction element; and the reaction chamber is not in fluidiccommunication with the nucleic acid extraction element when the wastestorage chamber is in fluidic communication with the reaction chamber,that is, the reaction chamber is selectively in fluidic communicationwith the nucleic acid extraction element or in fluidic communicationwith the waste storage chamber when appropriate.

Specifically, in one embodiment, a piston 7 is movably placed in thereaction chamber. When the piston moves up along the reaction chamber,the reaction chamber expands in volume, and its pressure decreases, thefluid is sucked into the reaction chamber from the nucleic acidextraction element or the waste storage chamber; when the fluid in thereaction chamber needs to be discharged, the piston moves down along thereaction chamber, the reaction chamber is shrank in volume, and itspressure increases, the fluid is pressed into the nucleic acidextraction element or the waste storage chamber from the reactionchamber.

In a preferred embodiment, the fluid flows from the reaction chamber tothe waste storage chamber under a negative pressure by reducing the airpressure in the waste storage chamber rather than pressurizing thereaction chamber by a piston. In this embodiment, the negative pressuredrives the fluid from the reaction chamber into the waste storagechamber, and the piston moves downward along the reaction chamber underthe negative pressure to prepare for the generation of negative pressurein the reaction chamber.

In some specific circumstances, for example, in order to further mix thefluid evenly, by reducing the pressure of the waste storage chamber, thefluid flows from the reaction chamber into the waste storage chamberunder the negative pressure, and then by reducing the pressure of thereaction chamber, the fluid flows from the waste storage chamber intothe reaction chamber under the negative pressure, by repeating thisprocess, the fluid is repeatedly exchanged between the two chambers tomix well. Wherein, the negative pressure of the reaction chamber isgenerated by the upward movement of the piston.

In a preferred embodiment, as shown in FIG. 1, the air pressure in thewaste storage chamber is reduced via a venting element that is incommunication with the waste storage chamber. Preferably, the ventingelement is a columnar suction column that communicates with the wastestorage chamber, more preferably it is a cylindrical suction column 6.As shown in FIGS. 5A and 5B, the suction column 6 is a hollow cylinderhaving one end communicated with the waste storage chamber 202. In apreferred embodiment, a gasket 601 is provided at the junction of thesuction column and the waste storage chamber 202 to improve the sealingof the suction column and the waste storage chamber, and the gasket ispreferably an elastic material. The other end of the suction column isexposed outside the nucleic acid extraction apparatus for connection toan air pumping device, preferably, the suction column external end 602is connected to the air pumping device via a hose. The air pumpingdevice described herein is preferably a vacuum pump or a negativepressure pump that reduces the air pressure in the waste storage chamber202 by evacuating the waste storage chamber 202. As the air pressure ofthe waste storage chamber 202 decreases, the fluid in the reactionchamber 201 enters the waste storage chamber 202, at this time, thefluid can be discharged from the reaction chamber 201 into the wastestorage chamber 202 without pressurizing the reaction chamber 201, whichis easy to operate and labor-saving. In a specific embodiment, thepiston 7 connected to the reaction chamber 201 moves downward relativeto the reaction chamber as the liquid in the reaction chamber isdischarged. The apparatus of this embodiment not only facilitates thedischarge of waste liquid from the reaction chamber 201 to the wastestorage chamber 202, but also is more suitable for the treatment ofbiological samples having certain infectivity or toxicity. Because thesuction column 6 is directly connected to the air pumping device duringthe process of nucleic acid extraction, the liquid flow can be achievedby vacuum pumping, and no waste gas will be discharged from the wastestorage chamber 202.

In a preferred embodiment, a solid phase material for nucleic acidextraction is provided in the reaction chamber 201 for capturing targetnucleic acids in the samples. In a preferred embodiment, the solid phasematerial for nucleic acid extraction is a magnetic bead 10. When thesolid phase material for nucleic acid extraction is a magnetic bead, thereaction chamber has a permanent magnet 11 inside so that the magneticbead is fixed in the reaction chamber. In a preferred embodiment, thereaction chamber 201 is a cylindrical chamber, and a hollow receivingchamber 2011 for accommodating the permanent magnet 11 is disposed inthe center of the reaction chamber, and the receiving chamber 2011 isalso a cylindrical chamber, the permanent magnet 11 is disposed in thehollow receiving chamber 2011. When the permanent magnet is placed inthe receiving chamber 2011, the magnetic bead 10 is fixed on the outersidewall of the receiving chamber 2011 by the adsorption of thepermanent magnet 11 (as shown in FIG. 1A). In a preferred embodiment,the bottom of the receiving chamber 2011 has a protrusion 8, and apermanent magnet 11 is placed on the protrusion 8. The setting of theprotrusion 8 enables the permanent magnet 11 to be disposed at anappropriate height, thereby ensuring that the magnetic bead 10 isadsorbed in an appropriate position on the sidewall of the receivingchamber 2011 rather than close to the bottom of the reaction chamber. Ina preferred embodiment, the piston 7 is a hollow piston having acylindrical shape. When the piston is placed in the reaction chamber201, it can be moved up and down along the reaction chamber or up anddown along the receiving chamber 2011. In a preferred embodiment, arecess 701 is provided on the sidewall at the bottom of the piston 7,that is, the sidewall at the bottom of the piston protrudes outwardly toform a recess 701, and the recess 701 and the sidewall at the bottom ofthe receiving chamber 2011 form a space for accommodating the magneticbead, as shown in the dotted line box of FIG. 1B. The magnetic beadbinds to free DNAs in the samples specifically to form a magneticbead-DNA complex, thereby immobilizing DNAs at a specific position inthe reaction chamber.

The samples include at least one of the following: cells, spores,microorganisms, biological tissues, biological fluids or environmentalsamples, etc.

In a preferred embodiment, the nucleic acid extraction elementcomprises: a lysis chamber 203 for adding and storing a mixture ofsample and lysate; a wash chamber 204 for adding and storing washingliquid; an eluent chamber 205 for adding and storing eluent; thereaction chamber 201 is sequentially in communication with the lysischamber 203, the wash chamber 204, and the eluent chamber 205 to extractand purify nucleic acids in the samples.

The lysate includes at least one of the following: guanidinehydrochloride, chaotropic salt, erythrocyte lysis reagent, chelatingagent, sodium hydroxide, DNase inhibitor, RNase inhibitor,anticoagulant, coagulant, protease, surfactant, etc.; the washing liquidincludes at least one of the following: ethanol, sodium chloride, Trishydrochloric acid, etc.; the eluent is sterile deionized water or Trishydrochloric acid. The specific flow is as follows: (1) the lysischamber 203 is in communication with the reaction chamber 201, andsamples and lysate enter the reaction chamber 201 for the lyticreaction, during the process, one the one hand, an additional devicesuch as a sonic vibrator can be connected to the apparatus of thepresent invention to facilitate the lysis of samples; and on the otherhand, after the mixture of lysate and samples enters the reactionchamber 201, the reaction chamber 201 is in communication with the wastestorage chamber 202, and the mixture of lysate and samples are exchangedrepeatedly between the reaction chamber and the waste storage chamber bychanging the pressure between the reaction chamber and the waste storagechamber, to facilitate the complete lysis of samples; (2) After thecompletion of the lytic reaction, the nucleic acids in the sample arebound to the magnetic bead 10 and immobilized in the reaction chamber201, and the lytic waste liquid enters the waste storage chamber 202;(3) the wash chamber 204 is in communication with the reaction chamber201, and washing liquid enters the reaction chamber 201, such that themagnetic bead that has been bound to the nucleic acid is immersed in thewashing liquid to clean and remove the impurities. During the process,on the one hand, the sound wave can be used to promote the mixing, andon the other hand, the washing liquid can be exchanged repeatedlybetween the reaction chamber 201 and the wash chamber 204 by moving upand down the piston, to ensure thorough washing; (4) the reactionchamber 201 is in communication with the waste storage chamber 202, thewaste storage chamber 202 is decompressed, the waste liquid isdischarged into the waste storage chamber 202, and the nucleic acid isbound to the magnetic bead and immobilized in the reaction chamber 201,and the washing process can be repeated several times according to theactual situation; (5) the eluent chamber 205 is in communication withthe reaction chamber 201, the eluent enters the reaction chamber 201, sothat the magnetic bead bound to nucleic acids is immersed in the eluent,the nucleic acid is separated from the magnetic bead and dissolved inthe eluent. In the process, the sound wave vibration can be used tofacilitate mixing and accelerate the elution, or the eluent can berepeatedly exchanged between the reaction chamber 201 and eluent chamber205 by moving up and down the piston, to ensure thorough elution.

In a preferred embodiment, after the nucleic acid is detached from themagnetic bead, the extraction of the nucleic acid in the sample iscompleted, at this time, the magnetic bead can be washed for the nexttime of extraction of nucleic acids, that is, the apparatus of thepresent invention can be repeatedly used for multiple times, to saveexperimental costs.

In a preferred embodiment, as shown in FIG. 4, the wash chamber 204comprises a primary wash chamber 2041, a secondary wash chamber 2042 anda tertiary wash chamber 2043. The primary wash chamber 2041, thesecondary wash chamber 2042, and the tertiary wash chamber 2043 aresequentially in communication with the reaction chamber 201,respectively. It is to be understood that the apparatus described hereinmay comprise a plurality of wash chambers, for example, two, three,four, etc., to satisfy nucleic acid purification using a plurality ofdifferent washing liquids, or to use a larger amount of the same washingliquid to perform washing, to improve the purity of nucleic acids.

In a preferred embodiment, the lysis chamber 203, the wash chamber 204,the eluent chamber 205 and the waste storage chamber 202 aresequentially in communication with the reaction chamber 201 through aplurality of mutually independent microfluidic channels, respectively.As shown in FIG. 3, FIG. 4, and FIG. 6, the lysis chamber 203, the washchamber 204, the eluent chamber 205, the waste storage chamber 202 andthe reaction chamber 201 are disposed in a cylindrical shell 2, whereinthe reaction chamber 201 is disposed in the center of inner circle of acylindrical shell, and the lysis chamber 203, the wash chamber 204, theeluent chamber 205 and the waste storage chamber 202 are distributed onthe outer circle of the cylindrical shell along the circumference of thereaction chamber 201, and are radially connected to the reaction chamber201; the plurality of mutually independent microfluidic channels aredisposed on a rotary disk 3, and the rotary disk is composed of twoparts; the shell 2 including the plurality of chambers is connected tothe rotary disk 3, and the rotation of the rotary disk 3 enables thereaction chamber 201 to pass through the plurality of microfluidicchannels to achieve fluidic communication with the lysis chamber 203,the wash chamber 204, the eluent chamber 205 or the waste storagechamber 202 in sequence.

As shown from FIG. 4, FIG. 6 and FIG. 7, the bottoms of the lysischamber 203, the wash chamber 204, the eluent chamber 205 and the wastestorage chamber 202 are provided with a through hole 207 respectively,and the bottom of the reaction chamber 201 is provided with a pluralityof through holes 207 corresponding to the through holes 207 at thebottoms of the above chambers one to one respectively. Through theone-to-one corresponding two through holes 207, the lysis chamber 203,the wash chamber 204, the eluent chamber 205, the waste storage chamber202 are in communication with the reaction chamber. As shown from FIG.7, the distances between two through holes that communicate the lysischamber 203 with the reaction chamber 201, communicate the primary washchamber 2041 with the reaction chamber 201, communicate the secondarywash chamber 2042 with the reaction chamber 201, communicate thetertiary wash chamber 2043 with the reaction chamber 201, communicatethe eluent chamber 205 with the reaction chamber 201 are equal, andthese through holes are distributed with equal distance from the centerof the reaction chamber. The distance between the two through holes thatcommunicate the waste storage chamber 202 with the reaction chamber 201is different from the distance between the two through holes thatcommunicate the above chambers and the reaction chamber.

FIG. 9 shows that a plurality of radially-extending independentmicrofluidic channels are distributed on the rotary disk, wherein eachmicrofluidic channel has two opening 306 on the outer surface of therotary disk, and the two opening 306 are respectively corresponding tothe two through holes 207 that communicate the lysis chamber with thereaction chamber, communicate the wash chamber with the reactionchamber, communicate the eluent chamber with the reaction chamber andcommunicate the waste storage chamber with the reaction chamber. Whenthe rotary disk is rotated horizontally by a certain angle, two openings306 of a microfluidic channel are engaged with the two through holes 207on the shell, to achieve fluidic communication between the abovechambers and the reaction chamber. The microfluidic channel includes aninlet microfluidic channel 301 and a plurality of outlet microfluidicchannels. In a preferred embodiment, there are four outlet microfluidicchannels, which are respectively named as a first outlet microfluidicchannel 302, a second outlet microfluidic channel 303, a third outletmicrofluidic channel and a fourth outlet microfluidic channel 305. Theoutlet microfluidic channels 302, 303, 304, and 305 have the samelength, but they differ from the inlet microfluidic channel 301 inlength. It is to be understood that the number of outlet microfluidicchannels can be set according to the number of times that the wasteliquid needs to be discharged.

In a preferred embodiment, the length of the inlet microfluidic channel301 is equal to the distance between two through holes that communicatethe lysis chamber 203 with the reaction chamber 201, communicate theprimary wash chamber 2041 with the reaction chamber 201, communicate thesecondary wash chamber 2042 with the reaction chamber 201, communicatethe tertiary wash chamber 2043 with the reaction chamber 201 and,communicate the eluent chamber 205 with the reaction chamber 201. Thelength of the outlet microfluidic channels 302, 303, 304, 305 is equalto the distance between the two through holes that communicate the wastestorage chamber 202 with the reaction chamber 201. Through the rotationof the rotary disk, the one inlet microfluidic channel 301 makes thelysis chamber 203, the primary wash chamber 2041, the secondary washchamber 2042, the tertiary wash chamber 2043 and the eluent chamber 205to be in communication with the reaction chamber 201 sequentially, whilethe waste storage chamber 202 is in communication with the reactionchamber 201 sequentially via the outlet microfluidic channels 302, 303,304, 305.

In a preferred embodiment, five groups of through holes that thatcommunicate the lysis chamber 203 with the reaction chamber 201,communicate the primary wash chamber 2041 with the reaction chamber 201,communicate the secondary wash chamber 2042 with the reaction chamber201, communicate the tertiary wash chamber 2043 with the reactionchamber 201 and, communicate the eluent chamber 205 with the reactionchamber 201 are equidistantly distributed around the center of thereaction chamber 201, that is, the radial extension lines of the fivegroups of through holes intersect at the center of the reaction chamber,and the angles formed by the radial extension lines of the five groupsof through holes are equal, preferably the angle is 36 degrees (as shownin FIG. 7).

The foregoing microfluidic channels are radially distributed with thecenter of the rotary disk 3 as a center of a circle, wherein the radialextension lines of the outlet microfluidic channels 302, 303, 304, and305 intersect at the center of the rotary disk 3, and the angles formedbetween the radially-extending lines are equal, preferably 36 degrees,wherein the center of the reaction chamber of the apparatus isoverlapped with the center of the rotary disk.

When the nucleic acid extraction apparatus is in a non-fluidiccommunication state (FIG. 8), the angle between the radial extensionline of the inlet microfluidic channel 301 and the radial extension lineof the two through holes that communicate the lysis chamber 203 and thereaction chamber 201 is 18 degrees. By horizontally rotating the rotarydisk 18 degrees clockwise relative to the lysis chamber 203, the lysischamber 203 can be in communication with the reaction chamber 201 viathe inlet microfluidic channel 301. By further horizontally rotating therotary disk clockwise, the waste storage chamber 202, the wash chamber204 or the eluent chamber 205 are in fluidic communication with thereaction chamber 201 sequentially.

In a preferred embodiment, the nucleic acid extraction apparatus furthercomprises a nucleic acid amplification element, and the nucleic acidamplification element comprises a PCR reaction solution chamber 206 thatis in communication with the reaction chamber, inside of which isprovided with reagents for a PCR reaction.

The PCR reaction reagents include at least one of the following: Bstpolymerase, Taq polymerase, reverse transcriptase, dNTPs, primers,probes, and the PCR reaction reagent is preferably a liquid.

In a preferred embodiment, as shown in FIG. 6, the PCR reaction solutionchamber 206 includes a primary PCR reaction solution chamber 2061 and asecondary PCR reaction solution chamber 2062, which are independent ofeach other. The primary PCR reaction solution chamber 2061 is incommunication with the reaction chamber 201 via the inlet microfluidicchannel 301, and the nucleic acids are eluted and dissolved in theeluent, at this time, the reaction chamber 201 is in communication withthe primary PCR reaction solution chamber 2061 to further move thepiston upward, and the PCR reaction solution enters the reaction chamber201 to mix with nucleic acids. Further, the PCR reaction solution andthe nucleic acids can be sufficiently mixed by sound wave oscillation.In this process, it is necessary to ensure that some of air is inhaledin the PCR reaction solution. The purpose of air inhalation is to ensurethat the PCR reaction solution enters the reaction chamber 201 in whole.

In a preferred embodiment, the nucleic acid amplification elementfurther comprises a PCR reaction tube 5 connected to the PCR reactionsolution chamber 206. The entire PCR reaction and the correspondingoptical result detection are completed in a PCR reaction tube 5. The PCRreaction tube 5 can be directly placed in a temperature controlinstrument for PCR amplification reaction or can be removed from anucleic acid amplification device and placed in a temperature controlinstrument for amplification reaction.

In a preferred embodiment, an input port 208 is provided at the bottomof the sidewall of the primary PCR reaction solution chamber 2061, andthe input port 208 is disposed on a sidewall having a certain heightdifference from the bottom of the primary PCR reaction solution chamber2061. A discharge port 209 is provided at the top of the sidewall of thesecondary PCR reaction solution chamber (FIG. 11), and the height of thedischarge port 209 from the bottom of the chamber is higher than theheight of the input port 208 from the bottom of the chamber. Both theinput port 208 and the discharge port 209 are through holes, and theinput port 208 and the discharge port 209 correspond to the first port501 and the second port 502 (FIG. 12) of the PCR reaction tube injectionchannel, respectively. The PCR reaction tube is in fluidic and gaseouscommunication with the PCR reaction solution chamber through the inputport 208, the discharge port 209, the first port 501 and the second port502 described above.

The piston 7 moves downward to pressurize the reaction chamber 201, andthe mixture of the PCR reaction solution and the nucleic acids entersthe PCR reaction solution chamber 206 from the reaction chamber 201 viathe inlet microfluidic channel 301. When the liquid level of the mixedsolution in the PCR reaction solution chamber 206 exceeds the input port208, the air at the top of the PCR reaction solution chamber 206 beginsto compress, causing the mixed solution to enter the PCR reaction tube 5from the input port 208. As the liquid enters, the air in the PCRreaction tube 5 is discharged from the discharge port 209, such that theliquid can enter the PCR reaction tube smoothly.

The discharge port has the following two functions: first, to evacuatethe air in the PCR reaction tube 5, to facilitate the entry of liquidinto the PCR reaction tube 5; second, to indicate whether the liquid issufficiently filled with the PCR reaction tube 5, and when there isliquid spill from the discharge port 209, it indicates that the mixedsolution is completely filled in the PCR reaction tube 5.

In a preferred embodiment, the input port 208 and the discharge port 209are two small holes having a pore size less than that of the first port501 and the second port 502 of the PCR reaction tube. By pressurizingthe reaction chamber 201, the mixed solution is pressed into the PCRreaction tube from the input port 208. This structure used caneffectively avoid the generation of bubbles in the mixed solutioninjected into the PCR reaction tube or facilitate the discharge ofbubbles, to prevent introduction of bubbles in the mixed solution thatmay interfere with the PCR reaction results.

In a preferred embodiment, a seal 9 is provided at the junction of thePCR reaction tube 5 and the PCR reaction solution chamber 206,preferably the seal 9 is made of an elastic material such as a silicagel. As shown in FIGS. 14A and 14B, seal 9 is a rectangular columnarstructure, with the upper and lower through holes penetrating thecylinder. Wherein, one side of the seal 9 is used for connection withthe PCR reaction tube 5, and the other side is used for connection withthe PCR reaction solution chamber 206. The two through holes 901 and 902on the connection surface of the PCR reaction tube 5 correspond to thefirst port 501 and the second port 502 of the PCR reaction tube 5,respectively, and the pore size thereof corresponds to the first port501 and the second port 502 of the PCR reaction tube. The first port 501and the second port 502 of the PCR reaction tube 5 are inserted into thethrough holes 901 and 902, respectively and are in interference fit withthe seal 9. The two through holes 903 and 904 on the connection surfaceof the PCR reaction solution chamber 206 correspond to the input port208 and the discharge port 209 of the CR reaction solution chamber 206respectively, and the pore size thereof corresponds to the input port208 and the discharge port 209. Further, there is a groove 905 at thejunction of the through holes 901 and 903 or through holes 902 and 904,and the groove 905 makes the input port 208 and the first port 501, orthe discharge port 209 and the second port 502 in a straight line. Thefluid enters from the through hole 903, flows through the groove 905 andthe first port 501, and then enters the injection channel of the PCRreaction tube 5, thereby entering the PCR reaction tube 5.

In a preferred embodiment, a sponge-like non-absorbent material capableof slowly leaking gas is provided at the top of the primary PCR reactionsolution chamber 2061, preferably the material is high-densityhydrophobic cotton. The high density hydrophobic cotton is set toachieve slow pumping of air, so that the air pressure of the primary PCRreaction solution chamber 2061 is kept consistent with that of thesecondary PCR reaction solution chamber 2062, to prevent the mixedsolution from flowing in the primary PCR reaction solution chamber 2061and avoid the generation of bubbles in the fluid. Specifically, thepiston moves downward to pressurize the reaction chamber 201, and themixed solution of the nucleic acids and the PCR reaction solution entersthe primary PCR reaction solution chamber 2061 from the reaction chamber201 via the inlet microfluidic channel 302, and the liquid level in theprimary PCR reaction solution chamber 2061 continuously rises, and whenthe liquid level is higher than the height of the input port, theprimary PCR reaction solution chamber 2061 starts to be pressurized. Atthis time, the air pressure of the secondary PCR reaction solutionchamber 2062 is atmospheric pressure. By setting the high densityhydrophobic cotton on the top of the primary PCR reaction solutionchamber 2061, the primary PCR reaction solution chamber 2061 can achieveslow venting such that the air pressure of the primary PCR reactionsolution chamber 2061 is consistent with the secondary PCR reactionsolution chamber 2062, making it easier for the liquid to enter the PCRreaction tube 5. In a preferred embodiment, the venting time of the highdensity hydrophobic cotton is set to 5 to 10 seconds. Preferably, thehigh density hydrophobic cotton is filled at ⅓ of the height of the bodyof the primary PCR reaction solution chamber 2061, for example, fixedlyfilled at the top of the primary PCR reaction solution chamber in a formof plunger piston. The high-density hydrophobic cotton does not absorbwater and does not absorb the PCR reaction solution, so that it will notproduce any influence on the nucleic acid extraction process.

In a preferred embodiment, as shown in FIG. 6, a rib 210 is disposed onthe wall of the eluent chamber 205 for reducing the amount of eluentused.

In a preferred embodiment, as shown in FIG. 13, a card slot 401 forfixing the PCR reaction tube 5 is provided on the base 4 of theapparatus of the present invention, and the card slot 401 is integrallyformed with the base 4, and the card slot 401 is perpendicular to thebase 4, and when the shell 2 is fixed on the base 4, the card slot 401is in contact with and longitudinally parallel to the shell 2.Specifically, the card slot 401 is fixedly connected to the outer wallof the PCR reaction solution chamber 206. The left and right sides ofthe card slot 401 are provided with grooves 402 for accommodating a PCRreaction tube, and the PCR reaction tube 5 is inserted and fixed in thegroove 402 of the card slot 401, such that the PCR reaction tube 5 andthe shell 2 are detachably connected.

In a preferred embodiment, as shown in FIG. 15, a boss 211 is disposedat the connection portion of the bottom of the shell 2 and the rotarydisk 3, to enhance the sealing effect of the shell 2 and the rotary disk3; and a boss 307 is provided at the connection portion of the rotarydisk 3 and the base 4, to reduce the friction when the rotary diskrotates.

FIG. 16 shows a schematic structural view of another embodiment of anucleic acid extraction apparatus of the present invention. In thisembodiment, a suction column 6 is not provided in the waste storagechamber 202. In this embodiment, by moving the piston 7 upwardly anddownwardly, the liquid exchange between the reaction chamber 201 and thelysis chamber 203, between the reaction chamber 201 and the wash chamber204, between the reaction chamber 201 and the eluent chamber 205,between the reaction chamber 201 and the waste storage chamber 202, thereaction chamber 201 and the PCR reaction solution chamber 206 can beachieved. Specifically, when moving the piston 7 upwardly, the pressureof the reaction chamber 201 decreases, the liquid is sucked into thereaction chamber 201, and when moving the piston 7 downwardly, thepressure of the reaction chamber 201 increases, and the liquid isdischarged into the reaction chamber 201. In this embodiment, anaperture is provided at the top cover 1 of the corresponding wastestorage chamber 202, and the aperture can ensure that liquid can besmoothly forced from the reaction chamber 201 into the waste storagechamber 202 when the reaction chamber 201 is pressurized. The nucleicacid extraction apparatus of this embodiment is particularly suitablefor extraction of nucleic acids from highly safe biological samples.

Example 2

In this embodiment, a method for extraction of nucleic acids using thenucleic acid extraction apparatus described in the Example 1 isdescribed.

Step 1: Open the lysis chamber 203, add a liquid sample, and mix thesample with lysate;

Step 2: rotate the rotary disk 3 horizontally by 18 degrees clockwise,so that the lysis chamber 203 communicates with the reaction chamber 201via the inlet microfluidic channel 301. Apply an external force to pushthe reaction chamber piston 7 upward, so that the mixed solution of thelysate and the sample enters the reaction chamber 201, and after lysisof samples, the released nucleic acids bind to the magnetic bead 10 inthe reaction chamber;

Step 3: rotate the rotary disk 3 horizontally by 18 degrees clockwise,so that the waste storage chamber 202 communicates with the reactionchamber 201 via the first outlet microfluidic channel 302. Connect thevacuum pump to the suction column 6 for evacuation, and the lytic wasteliquid enters the waste storage chamber 202, the magnetic beads bound tonucleic acids are immobilized in the reaction chamber 201;

Step 4: rotate the rotary disk 3 horizontally by 18 degrees clockwise,so that the primary wash chamber 2041 communicates with the reactionchamber 201 via the inlet microfluidic channel 301. Apply an externalforce to push the reaction chamber piston 7 upward, and the washingliquid enters the reaction chamber 201, such that the magnetic beadsbound to nucleic acids are immersed into the washing liquid, to wash andremove impurities;

Step 5: rotate the rotary disk 3 horizontally by 18 degrees clockwise,so that the waste storage chamber 202 communicates with the reactionchamber 201 via the second outlet microfluidic channel 303. Connect thevacuum pump to the suction column 6 for evacuation, and the washingwaste liquid enters the waste storage chamber 202, the magnetic beadsbound to nucleic acids are immobilized in the reaction chamber 201;

Step 6: rotate the rotary disk 3 horizontally by 18 degrees clockwise,so that the secondary wash chamber 2042 communicates with the reactionchamber 201 via the inlet microfluidic channel 301. Apply an externalforce to push the reaction chamber piston 7 upward, and the washingliquid enters the reaction chamber 201, such that the magnetic beadsbound to nucleic acids are immersed into the washing liquid, to wash andremove impurities;

Step 7: rotate the rotary disk 3 horizontally by 18 degrees clockwise,so that the waste storage chamber 202 communicates with the reactionchamber 201 via the third outlet microfluidic channel 304. Connect thevacuum pump to the suction column 6 for evacuation, and the washingwaste liquid enters the waste storage chamber 202, the magnetic beadsbound to nucleic acids are immobilized in the reaction chamber 201;

Step 8: rotate the rotary disk 3 horizontally by 18 degrees clockwise,so that the tertiary wash chamber 2043 communicates with the reactionchamber 201 via the inlet microfluidic channel 301. Apply an externalforce to push the reaction chamber piston 7 upward, and the washingliquid enters the reaction chamber 201, such that the magnetic beadsbound to nucleic acids are immersed into the washing liquid, to wash andremove impurities;

Step 9: rotate the rotary disk 3 horizontally by 18 degrees clockwise,so that the waste storage chamber 202 communicates with the reactionchamber 201 via the fourth outlet microfluidic channel 305. Connect thevacuum pump to the suction column 6 for evacuation, and the washingwaste liquid enters the waste storage chamber 202, the magnetic beadsbound to nucleic acids are immobilized in the reaction chamber 201;

Step 10: rotate the rotary disk 3 horizontally by 18 degrees clockwise,so that the eluent chamber 205 communicates with the reaction chamber201 via the inlet microfluidic channel 301. Apply an external force topush the reaction chamber piston 7 upward, and the eluent enters thereaction chamber 201, such that the magnetic beads bound to nucleicacids are immersed into the eluent, and the nucleic acids are detachedfrom the magnetic beads and dissolved in the eluent;

Step 11: rotate the rotary disk 3 horizontally by 36 degrees clockwise,so that the primary PCR reaction solution chamber 2061 communicates withthe reaction chamber 201 via the inlet microfluidic channel 301. Applyan external force to push the reaction chamber piston 7 upward, and thePCR reaction solution enters the reaction chamber 201, to mix thenucleic acid dissolved in the eluent with a PCR reaction reagent;

Step 12: Apply an external force to push the reaction chamber piston 7downward, so that the mixed solution in the reaction chamber 201 entersthe primary PCR reaction solution chamber 2061 via the inletmicrofluidic channel 301. When the liquid level of the mixed solution inthe primary PCR reaction solution chamber 2061 is higher than the inputport 208 of the primary PCR reaction solution chamber 2061, the mixedsolution enters the PCR reaction tube 5 via the input port 208 until themixed solution overflows from the discharge port 209 of the secondaryPCR reaction solution chamber 2062, to complete the liquid inlet of PCRreaction tube 5;

Step 13: Place the PCR reaction tube 5 in a temperature control device,and carry out nucleic acid fluorescent PCR amplification or fluorescentisothermal amplification according to the established PCR amplificationprogram.

In addition, the embodiments described in the following paragraphs arealso a part of the present invention.

1. A nucleic acid extraction apparatus, comprising:

a nucleic acid extraction element, a waste storage chamber, and areaction chamber, wherein the reaction chamber is selectively incommunication with the nucleic acid extraction element and the wastestorage chamber, and the apparatus realizes fluid exchange through thepressure between the waste storage chamber and the reaction chamber.

2. The apparatus according to paragraph 1, wherein for the apparatus,fluid enters the waste storage chamber from the reaction chamber byreducing the air pressure of the waste storage chamber.

3. The apparatus according to paragraph 2, the air pressure of the wastestorage chamber for the apparatus is reduced by a venting element thatis in communication with the waste storage chamber.

4. The apparatus according to paragraph 3, wherein the venting elementis a columnar suction column.

5. The apparatus according to paragraph 4, wherein the venting elementis a cylindrical suction column.

6. The apparatus according to paragraph 5, wherein a gasket is disposedat the junction of the suction column and the waste liquid storagechamber.

7. The apparatus according to paragraph 1, wherein a solid phasematerial for nucleic acid extraction is disposed in the reactionchamber.

8. The apparatus according to paragraph 7, wherein the solid phasematerial for nucleic acid extraction is a magnetic bead.

9. The apparatus according to paragraph 1, wherein the nucleic acidextraction element comprises:

a lysis chamber for adding and storing a mixture of sample and lysate;or,

a wash chamber for adding and storing washing liquid; or,

an eluent chamber for adding and storing eluent;

wherein, one or more of the lysis chamber, the wash chamber or theeluent chamber are in fluidic communication with the reaction chamber,respectively.

10. The apparatus according to paragraph 9, wherein the wash chambercomprises a primary wash chamber, a secondary wash chamber and atertiary wash chamber, one or more of the primary wash chamber, thesecondary wash chamber or the tertiary wash chamber being in fluidiccommunication with the reaction chamber, respectively.

11. The apparatus according to paragraph 1, wherein the apparatus mayfurther comprise a nucleic acid amplification element, and the nucleicacid amplification element is a PCR reaction solution chamber that is influidic communication with the reaction chamber, inside of which isprovided with reagents for a PCR reaction.

12. The apparatus according to paragraph 11, wherein the nucleic acidamplification element further comprises a PCR reaction tube that is influidic communication with the PCR reaction solution chamber.

13. The apparatus according to paragraph 12, wherein a sidewall of thePCR reaction solution chamber is provided with an input port and adischarge port that are in fluidic communication with the PCR reactiontube.

14. The apparatus according to paragraph 13, wherein the PCR reactionsolution chamber comprises a primary PCR reaction solution chamber and asecondary PCR reaction solution chamber, wherein the primary PCRreaction solution chamber is in fluidic communication with the reactionchamber, wherein an input port is disposed at the bottom of the sidewallof the primary PCR reaction solution chamber, and a discharge portdisposed at the top of the sidewall of the secondary PCR reactionsolution chamber.

15. The apparatus according to any one of paragraphs 1 to 14, whereinthe apparatus comprises a microfluidic channel for fluidic communicationwith a reaction chamber, a waste storage chamber, a nucleic acidextraction element or a nucleic acid amplification element.

16. The apparatus according to paragraph 15, wherein the microfluidicchannel is radially extended on a rotary disk, and the reaction chamber,the waste storage chamber, the nucleic acid extraction element or thenucleic acid amplification element are in communication with or not incommunication with each other by the rotation of the rotary disk.

17. The apparatus according to paragraph 1, wherein the fluid exchangeis achieved by a microfluidic channel.

18. The apparatus according to paragraph 1, wherein the waste storagechamber is not in fluidic communication with the reaction chamber whenthe reaction chamber is in fluidic communication with the nucleic acidextraction element, and the reaction chamber is not in fluidiccommunication with the nucleic acid extraction element when the wastestorage chamber is in fluidic communication with the reaction chamber.

19. The apparatus according to paragraph 18, wherein the apparatuscomprises a piston, the fluid in the nucleic acid extraction elemententers the reaction chamber by the movement of the piston.

20. The apparatus according to paragraph 19, wherein the movement of thepiston is an upward movement along the reaction chamber.

21. The apparatus according to paragraph 20, wherein the apparatusfurther comprises a venting element that causes the waste storagechamber to generate a negative pressure, thereby allowing the fluid inthe reaction chamber to enter the waste storage chamber.

22. The apparatus according to paragraph 21, wherein the negativepressure drives the piston to move downwardly along the reactionchamber.

23. The apparatus according to paragraph 22, wherein the venting elementthat causes the waste storage chamber to generate a negative pressure isa suction column connected to the waste storage chamber.

24. A method for extracting a nucleic acid, comprising the followingsteps:

(1) providing a nucleic acid extraction apparatus, wherein the apparatuscomprising:

a nucleic acid extraction element for extraction of nucleic acids;

a waste storage chamber for storing waste liquid during the reaction;

a reaction chamber, being selectivity in communication with the nucleicacid extraction element and the waste storage chamber;

(2) allowing the reaction chamber to be in fluidic exchange with thewaste storage chamber or the nucleic acid extraction element to achieveextraction of nucleic acids.

25. The method according to paragraph 24, wherein the fluid is exchangedbetween the reaction chamber, the waste storage chamber, or the nucleicacid extraction element by a pressure change between the waste storagechamber and the reaction chamber.

26. The method according to paragraph 25, wherein the fluid is allowedto enter the waste storage chamber from the reaction chamber by reducingthe air pressure in the waste storage chamber.

27. The method according to paragraph 26, wherein the air pressure ofthe waste storage chamber is reduced by a venting element that is incommunication with the waste storage chamber.

28. The method according to paragraph 27, wherein the venting element isa cylindrical suction column.

29. The method according to paragraph 24, wherein A solid phase materialfor nucleic acid extraction is provided in the reaction chamber to bindthe nucleic acid in the sample to the solid phase material for nucleicacid extraction.

30. The method according to paragraph 29, wherein the solid phasematerial for nucleic acid extraction is a magnetic bead.

31. The method according to paragraph 24, wherein the nucleic acidextraction element comprises:

a lysis chamber for adding and storing a mixture of sample and lysate;or,

a wash chamber for adding and storing washing liquid; or,

an eluent chamber for adding and storing eluent;

wherein, one or more of the lysis chamber, the wash chamber or theeluent chamber are in fluidic communication with the reaction chamber,respectively.

32. The method according to paragraph 31, wherein the wash chambercomprises a primary wash chamber, a secondary wash chamber and atertiary wash chamber, one or more of the primary wash chamber, thesecondary wash chamber or the tertiary wash chamber being in fluidiccommunication with the reaction chamber, respectively.

33. The method according to paragraph 24, wherein the method furthercomprises a step of nucleic acid amplification, and the reaction chamberis in fluidic communication with the nucleic acid amplification elementto achieve nucleic acid amplification, wherein, the nucleic acidamplification element is a PCR reaction solution chamber that is influidic communication with the reaction chamber, inside of which isprovided with reagents for a PCR reaction.

34. The method according to paragraph 33, wherein the nucleic acidamplification element further comprises a PCR reaction tube that is influidic communication with the PCR reaction solution chamber, to allowfluid to enter the PCR reaction tube for amplification reaction.

35. The method according to paragraph 34, wherein the PCR reaction tubeachieves fluidic communication through an input port and a dischargeport disposed on sidewall of the PCR reaction solution chamber.

36. The method according to paragraph 35, wherein the PCR reactionsolution chamber comprises a primary PCR reaction solution chamber and asecondary PCR reaction solution chamber, the primary PCR reactionsolution chamber is in fluidic communication with the reaction chamber,wherein an input port is disposed at the bottom of the sidewall of theprimary PCR reaction solution chamber, and a discharge port disposed atthe top of the sidewall of the secondary PCR reaction solution chamber.

37. The method according to any one of paragraphs 24 to 36, whereinfluidic communication or exchange between the reaction chamber, thewaste storage chamber, the nucleic acid extraction element or thenucleic acid amplification element is achieved by a microfluidic channeldisposed on the nucleic acid extraction apparatus.

38. The method according to paragraph 37, wherein the microfluidicchannel is radially extended on a rotary disk, and the reaction chamber,the waste storage chamber, the nucleic acid extraction element or thenucleic acid amplification element are in communication with or not incommunication with each other by the rotation of the rotary disk.

39. The method according to paragraph 24, wherein the fluid exchange isachieved by a microfluidic channel.

40. The method according to paragraph 24, wherein the waste storagechamber is not in fluidic communication with the reaction chamber whenthe reaction chamber is in fluidic communication with the nucleic acidextraction element; and the reaction chamber is not in fluidiccommunication with the nucleic acid extraction element when the wastestorage chamber is in fluidic communication with the reaction chamber.

41. The method according to paragraph 40, wherein the fluid in thenucleic acid extraction element enters the reaction chamber by themovement of a piston disposed on the reaction chamber.

42. The method according to paragraph 41, wherein the movement of thepiston is an upward movement along the reaction chamber.

43. The method according to paragraph 42, wherein the fluid in thereaction chamber enters the waste storage chamber by a venting elementthat causes the waste storage chamber to generate a negative pressure.

44. The method according to paragraph 43, wherein the venting elementthat causes the waste storage chamber to generate a negative pressure isa suction column connected to the waste storage chamber and the negativepressure drives the piston to move downwardly along the reactionchamber.

The foregoing descriptions are merely the preferred embodiments of thepresent invention, and are not intended to limit the present invention.Any modifications, equivalents, improvements, etc. made withoutdeparting from the spirit and scope of the present invention shall fallinto the scope of protection of the present invention.

1. A nucleic acid extraction apparatus, comprising: a plurality ofnucleic acid extraction chambers; and a reaction chamber, wherein thereaction chamber is selectively in communication with one of the nucleicacid extraction chambers; a hollow piston disposed in the reactionchamber and configured to move between a first position and a secondposition in the reaction chamber to change a pressure in the reactionchamber; a magnetic bead disposed in the reaction chamber for binding anucleic acid; a receiving chamber for accommodating a permanent magnetas to fix the magnetic bead on an outer side wall of the receivingchamber; wherein the receiving chamber is disposed in the hollow piston,and the hollow piston is movable relative to the receiving chamber; andwherein a recess is provided on the sidewall at a bottom of the hollowpiston, and when the hollow piston is at the second position, the recessand the outside wall of the receiving chamber form a space foraccommodating the magnetic bead therein.
 2. The apparatus according toclaim 1, wherein the apparatus further comprise a nucleic acidamplification element and the nucleic acid amplification element is anucleic acid amplification chamber that is in fluidic communication withthe reaction chamber.
 3. The apparatus according to claim 2, wherein thenucleic acid amplification chamber is a PCR reaction tube.
 4. Theapparatus according to claim 1, wherein nucleic acid extraction chamberscomprise a lysis chamber with lysate reagent for receiving a sample; awash chamber for adding and storing washing liquid; an eluent chamberfor adding and storing eluent reagent.
 5. The apparatus according toclaim 4, wherein the reaction chamber selectively in communication withone or more of the lysis chamber, the wash chamber or the eluent chamberof the nucleic acid extraction chambers.
 6. The apparatus according toclaim 5, wherein the apparatus further comprises a microfluidic channelfor fluidic communication with the reaction chamber; the lysis chamber,the wash chamber or the eluent chamber of the nucleic acid extractionchambers.
 7. The apparatus according to claim 6, wherein themicrofluidic channel is radially extended on a rotary disk, and thereaction chamber is in communication with or not in communication withone or more of the lysis chamber, the wash chamber or the eluent chamberof the nucleic acid extraction chambers by the rotation of the rotarydisk.
 8. The apparatus according to claim 1, wherein the apparatusfurther comprise a rotary disk, the reaction chamber is in communicationwith or not in communication with one or more of the nucleic acidextraction chambers by the rotation of the rotary disk.
 9. The apparatusaccording to claim 8, wherein a microfluidic channel is disposed in therotary disk.
 10. The apparatus according to claim 1, wherein a rotarydisk disposed at the bottom of the shell, wherein the rotary diskincludes a microfluidic channel for fluidic communication with thereaction chamber and one of the nucleic acid extraction chambers. 11.The apparatus according to claim 10, wherein a fluid in the one of thenucleic acid extraction chambers enters into the reaction chamber by amovement of the piston.
 12. The apparatus according to claim 11, whereinthe movement of the piston is an upward movement along the receivingchamber.
 13. The apparatus according to claim 1, wherein the washchamber comprises a primary wash chamber, a secondary wash chamber and atertiary wash chamber, one or more of the primary wash chamber, thesecondary wash chamber or the tertiary wash chamber being in fluidiccommunication with the reaction chamber.
 14. The apparatus according toclaim 13, wherein the waste chamber is not in fluidic communication withthe reaction chamber when the reaction chamber is in fluidiccommunication with the lysis chamber and the reaction chamber is not influidic communication with the lysis chamber when the waste chamber isin fluidic communication with the reaction chamber.
 15. The apparatusaccording to claim 14, wherein a fluid in the lysis chamber enters thereaction chamber by a movement of the piston along the receiving chamber16. The apparatus according to claim 9, wherein the wash chambercomprises a primary wash chamber, a secondary wash chamber and atertiary wash chamber, one or more of the primary wash chamber, thesecondary wash chamber or the tertiary wash chamber being in fluidiccommunication with the reaction chamber, respectively.
 17. The apparatusaccording to claim 1, wherein the apparatus further comprises a nucleicacid amplification element comprising a PCR reaction tube that is influidic communication with a PCR reaction solution chamber.
 18. Theapparatus according to claim 17, wherein a sidewall of the PCR reactionsolution chamber is provided with an input port and a discharge portthat are in fluidic communication with the PCR reaction tube.
 19. Theapparatus according to claim 18, wherein the PCR reaction solutionchamber comprises a primary PCR reaction solution chamber and asecondary PCR reaction solution chamber, wherein the primary PCRreaction solution chamber is in fluidic communication with the reactionchamber, wherein an input port is disposed at the bottom of the sidewallof the primary PCR reaction solution chamber, and a discharge portdisposed at the top of the sidewall of the secondary PCR reactionsolution chamber.